Essential role of MD-2 in TLR4-dependent signaling during Helicobacter pylori- associated gastritis

Toll样受体4(Toll-like Receptor 4，TLR4)是模式识别受体中的一员，是内毒素的主要受体。MD-2在物理上与细胞表面的TLR4结合，并赋予内毒素反应性。幽门螺杆菌脂多糖是引起胃炎的主要毒力因子之一。我们在这里展示了MD-2在幽门螺杆菌相关性胃炎中TLR4依赖的信号转导中的作用。本研究采用幽门螺杆菌感染患者和未感染幽门螺杆菌患者及4株胃癌细胞株的胃活检标本。用RT-PCR检测TLR-4和MD-2在活检标本和细胞系中的表达。免疫组织化学方法检测TLR-4在组织切片中的定位。在体外功能检测中，我们建立了稳定表达TLR4和MD-2的AGS细胞。用流式细胞仪检测TLR4的细胞分布。用报告基因分析法检测NF-κB的活化及IL-8和MD-2启动子的激活。幽门螺杆菌感染可上调胃粘膜TLR4和MD-2的表达。TLR4染色主要见于上皮细胞，位于细胞质和根尖表面。在幽门螺杆菌脂多糖刺激下，细胞表面TLR4的表达显著增加，并增强了NF-κB和IL-8启动子的活性。活的幽门螺杆菌也刺激了MD-2的转录激活。本研究发现，在幽门螺杆菌感染过程中，胃上皮细胞中MD-2的表达增加，提示TLR4/MD-2系统是参与胃对幽门螺杆菌脂多糖应答的有效受体复合体。

Toll-like receptor 4(TLR4), a member of pattern recognition receptors, is the main receptor of lipopolysaccharide(LPS). MD-2 physically associates with TLR4 on the cell surface and confers LPS responsiveness. H.pylori LPS is one of the major virulence factors for induction of gastritis. We demonstrated here the role of MD-2 in TLR4-dependent signaling in H.pylori-associated gastritis. Gastric biopsy samples collected from patients with and without H.pylori infection and 4 gastric cancer cell lines were used for this study. TLR-4 and MD-2 expression in biopsy specimens and the cell lines was examined by using RT-PCR. Localization of TLR-4 in histological sections was evaluated by immunohistochemistry. For in vitro functional assays, we established stable transfectants of AGS cells expressing TLR4 and MD-2. Cellular distribution of TLR4 was examined by flow cytometry. NF-κB activation and activation of IL-8 and MD-2 promoters were assessed by reporter gene assay. H.pylori infection up-regulated the TLR4 and MD-2 expression in gastric mucosa. TLR4 staining was observed predominantly in epithelial cells, located in both the cytoplasm and at the apical surface. MD-2 transfection in AGS cells markedly increased cell surface expression of TLR4 and augmented the activation of NF-κB and IL-8 promoter upon stimulation with H.pylori LPS. Live H.pylori also stimulated transcriptional activation of MD-2. This study revealed that MD-2 expression is elevated in gastric epithelial cells during H.pylori infection, suggesting that the TLR4/MD-2 system is a potent receptor complex involved in the response to H.pylori LPS in the stomach.